The goal of this project is to manipulate the chemical properties of both the cell surface (through emerging metabolic substrate-based oligosaccharide engineering technology) and the material to which the cell binds. By using this approach, a highly-compatible adhesion interface can be engineered that supports novel modes of carbohydrate-based cell adhesion and - building on the intimate biochemical links between adhesion and signaling pathways - provides an innovative method to control stem cell fates. Specifically, this proposal is based on the "proof-of-principle" demonstration that the thiol-bearing N-acetylmannosamine (ManNAc) analog "Ac5ManNTGc" can intercept the sialic acid biosynthetic pathway, be converted into the corresponding thiol-bearing "Neu5TGc" form of sialic, and then be displayed in the oligosaccharides that cover the surfaces of mammalian cells. Once on the cell surface, Neu5TGc facilitates the attachment of the host cell to other thiol-bearing cells via trans-disulfide bonds as well as to chemically compatible maleimide- derivatized or gold surfaces. Importantly, cells gain new behaviors upon undergoing this novel mode of adhesion;in particular the Wnt signaling pathway was activated in human embryoid body-derived (hEBD) stem cells causing them to undergo differentiation to neurons. The first specific aim is focused on fine-tuning the chemical properties of the cell surface (by changing the thiol linker length to the underlying sugar residue, by changing Neu5GTc surface density, and by control of thiol redox chemistry) and material (by patterned, microfabricated gold and maleimide display) to determine the precise factors at the celhmaterial interface that contribute to activation of Wnt signaling. The second specific aim will test a multi-step model of the genetic, molecular and biochemical steps involved Ac5ManNTGc-activation of Wnt signaling. The first two aims, tightly focused on one set of surface chemistries (the thiohgold/maleimide interface) and one pathway (Wnt signaling), will provide detailed mechanistic information that will provide a solid scientific foundation for the addition of "small molecule oligosaccharide engineering" to the tissue engineering toolkit. This methodology is anticipated to be broadly applicable, and towards this end, the third specific aim (1) will evaluate the ability of thiol-bearing ManNAc analogs to activate the Wnt pathway and neural differentiation in a second embryonic stem cell line as well as in adult stem cells and (2) investigate the impact of thiohgold/maleimide interface on additional signaling pathways (G-protein, TGF-[unreadable], and JNK) implicated in the differentiation of neurons.